Transgenic R e s e a r c h 4, 149-150 (1995)

A simple method of DNA extraction from whole tissues

and blood using glass powder for detection of
transgenic animals by PCR

JOE ATTAL, MARCO CAJERO-JUAREZ and L O U I S - M A R I E

HOUDEBINE*
Unit~ de Differentiation Cellulaire, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas
Cedex, France (Fax: 331 34 65 22 41)

A very simple and reliable method to extract DNA directly from mouse tail, rabbit ear and blood is described.
Tissue was homogenized in a solution of NaI and the DNA was extracted using glass powder. The extracted DNA
was obtained in sufficient quantity and purity to allow direct detection of transgene by PCR.
Keywords: DNA extraction; glass powder; PCR; transgene

In~oducfion mixture was centrifuged at 10000 g for 1 min. The

resulting aqueous phase was saved and mixed with 8 gl of
The polymerase chain reaction (PCR) has proven to be an
glass powder suspension (Gene-Clean). The mixture was
extremely powerful tool for detecting a gene or a
shaken for 5 min at 4"C and centrifuged at 10000 g for
transgene within a genome. The preparation of DNA from
30 s. The supernatant was discarded and the pellet was
both tissue cells and blood often involve lengthy purific-
resuspended by vortexing in 300 ~tl of a wash solution
ation procedures (Maniatis et aI., 1982; Keller et aL,
(50 ml ethanol, 50 ml water containing: 20 mM Tris,
1988; Laird et al., 1991).
pH 7.4, 1 mM EDTA and 0.1 M NaC1). The glass powder
New simple extraction procedures for DNA purific-
was pelleted again by centrifuging for 30 s at 10000 g.
ation have been recently developed. Most of them use
The pellet was resuspended in 50 ~tl water. The mixture
blood cells as a source of DNA (Ishizawa et aL, 1991;
was incubated for 5 rain at 55"C and centrifuged at
Nordvag and Husby, 1992). Blood samples are not easily
10000 g for 1 min. The supernatant contained eluted
obtained in young animals and transgene detection has to
DNA.
be carried out from tissues (tail or ear). Recently, it has
The same protocol was used to extract DNA from
been shown that a direct PCR can be performed using
50 ~tl whole blood of older animals. Alternatively, crude
crude tissue extracts (Panaccio et aL, 1993). However,
nuclei were prepared and DNA extracted using the
special PCR conditions are required with this material
procedure described above.
(use of Tth polymerase instead of Taq polymerase,
samples cycled in 100% formamide prior to PCR) and the
amount of DNA analysed is not controlled. In the present Results
paper, a very fast and simple method of DNA extraction
The reJults shown in Fig. 1 indicate that the DNA
based on the use of glass powder is described.
obtained by the method depicted here was undegraded
and essentially free of contaminant material. Interestingly,
Materials and methods similar amounts of DNA were present in each lane. This
amount corresponded to 300 to 400 ng of DNA for 3 ~1
20 to 30 mg of excised tissue from newborn animals
of eluate in the case of both tail and ear extracts. The
(mouse tail, rabbit ear) were homogenized in 200 ~tl of
DNA concentration was essentially the same in the
6 M NaI, 1.5% sodium sulphite in a 1.5 ml Eppendorf
different samples; this is obviously due to the fact that the
tube using a pestle Pellet Treff (Poly-Labo 82.555). The
glass powder was saturated by the excess of DNA present
*To whom correspondence should be addressed. in the extracts. Hence, using the procedure depicted
0962-8819 9 1995 Chapman & Hall
150
Fig. 1. Examination of DNA fractions extracted from both
tissues and blood using glass powder. In all eases, 3 ~tl of the final
eluates were run on 1% agarose gel. 1, 2 and 3 extracts from
mouse tails; 4, 5 and 6 extracts from 50 ~tl whole blood; 7, 8 and
9 extracts from rabbit ears.
Fig. 2. Detection of a single copy gene from rabbit DNA extracts
by PCR. The samples were amplified with the following oligo-
primers:
5'TACTITATGAAATCTGGAAA3' and 5'AGCAAGGCIT-
GTTITGGTAG3'. Lane 1: amplification with a plasmid
containing the rabbit Ctsl-casein eDNA; lanes 2, 3 and 4:
amplification using 1, 2 and 3 ~tl of eluate obtained from whole
blood extract; lane 5: control amplification without DNA; lane 6,
7 and 8: amplification using 1, 2 and 3 Ixl of an ear extract; lane
9" phiX174 Hae III markers.
Fig. 3. Detection of transgenes by PCR of DNA from mice
whole blood and tails and from rabbit ears. In each case, 3 ~tl of
glass powder eluate were used for PCR. Lanes 1 and 2: whole
blood DNA from mice harbouring bGH eDNA; lane 3: whole
blood DNA from a control mouse; lanes 4 and 5: tail DNA from
2 mice harbouring the bGH eDNA; lane 6: tail DNA from a
control mouse; lane 7 and 9: ear DNA from 2 rabbits harbouring
the human erythropoietin gene; lane 8: ear DNA from a control
rabbit; lane 10:phiX174 Hae III markers.
above, the amount of D N A need not be measured in the
different samples prior to the PCR step. This interpreta-
tion was confirmed by the fact that D N A from blood
sample was less abundant. Indeed, 50 ~tl of whole blood
are expected to contain at most 2 ~tg of DNA. The 8 ~tl of
glass powder added to the extracts are expected to retain
not more than 7 ~tg of DNA. It is saturated in the case of
tissue extracts but not in the case of blood extract.
A t t a l et al.
In order to evaluate the quality of the D N A prepared
by the method described here, the detection of a single
copy gene was carried out using PCR to amplif~ a
sequence specific to Ctsl-casein gene (Jolivet et al., 1992).
Results shown in Fig. 2 reveal that the gene was easily
detected and in a reproducible manner.
In order to confirm that the method described above
can be used to detect transgenes, blood and tissue extracts
from both transgenic and control animals were used. D N A
from both whole blood and tail from transgenic mice
harbouring b G H cDNA was assayed to detect the trans-
gene. The mice which were shown to be transgenic using
the CTAB-DNA precipitation method (Gustincich et al.,
1991) also appeared to contain the b G H sequence when
using the method depicted here. Similarly, transgenic
rabbits known to harbour the human erythropoietin gene
were found to contain the transgene (Fig. 3).
The D N A extraction method using glass powder which
is described in the present paper is extremely rapid (less
than 20 min for one sample) and reliable. It may be used
for various kinds of samples as solid tissues, whole blood
and certainly cultured cells. Given its great simplicity, it
should be amenable to automation.
Acknowledgements
M. Cajero-Juarez is the recipient of a C O N A C Y T
fellowship (Mexico).
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